Following local trauma, damaged axons in the adult mammalian central nervous system (CNS) regress, and subsequent regeneration of these damaged axons is very limited. This is thought to strongly constrain recovery of CNS function and contributes to paralysis, sensory dysfunction and cognitive impairment. To date, the study of brain axon regeneration has almost exclusively relied upon postmortem analysis of fixed tissue from intact preparations, which yields static images. These snapshots are suboptimal for evaluating therapeutic interventions: They often fail to distinguish regenerating axons from sprouting of undamaged fibers or spared axons at the lesion site. We have developed a model system in which long-distance regeneration of axons can be studied with time-lapse imaging in the intact adult mouse brain. Systemic treatment of adult rats with p-chloro-amphetamine (PCA) causes rapid regression of dorsal raphe serotonin axons, followed by a slow return of serotonergic innervation over many weeks. We have adapted this PCA protocol to adult BAC transgenic mice in which the complete extent of serotonin neurons is labeled with EGFP. Using a two-photon microscope and a cranial window, we can repeatedly image the same volume of neocortex and thereby track serotonergic axons before and e 13 weeks after lesion with PCA to provide time-lapse measurements of identified surviving, regressing and regenerating fibers. Here, we propose to develop and extend this model system. Aim 1. Do rapid dynamic events in regenerating serotonin axons following lesion with PCA predict features of stable regenerated axon morphology? To date, we have performed a low temporal resolution survey with weekly measurements. This provides an overview but has not allowed for examination of axons on a minutes-to-days timescale. We propose to augment our dataset with measurements at 10 min and daily intervals during two crucial periods: immediately following PCA to capture regression and ~ 7-8 weeks following PCA, when many pioneering fibers are entering the field of view. Aim 2. What are the key short and long-term structural dynamics of regenerating 5HT axons following a thermal lesion of the neocortex? We propose to repeat in vivo time-lapse imaging of serotonin axons, replacing PCA treatment with focal thermal lesions. Our goal is to have two well-defined model systems for axonal damage and regeneration, one conventional, pan-cellular and glial-scar-forming and the other cell-type-specific and non- scar forming in order to compare molecular interventions and candidate therapies for functional recovery. Aim 3. Is expression of integrin 1 in serotonin neurons required for all phases of axonal regeneration in the neocortex following thermal or PCA lesion? Serotonin neurons express high levels of integrin 1, a protein that forms part of the receptor for the permissive growth substrate laminin. We shall cross mouse strains: floxed integrin 1 with serotonin transporter-driven Cre, to selectively delete integrin 1 in serotonin neurons coupled with in vivo time-lapse imaging of serotonin axons in response to PCA and thermal lesions. PUBLIC HEALTH RELEVANCE: Following stroke or traumatic brain injury in adults, recovery of function is often very limited, resulting in persistent paralysis, sensory dysfunction, cognitiv impairment and disorders of mood. Researchers have attempted to develop therapies to promote recovery of function by stimulating the regrowth of axons, the long information-sending extensions of nerve cells in the brain. We have developed a new system for making time- lapse images of regenerating axons in the brain of this mouse, with the ideas that this will provide an ideal test- bed for developing new therapeutic measures.